Methods, compositions, and kits for detecting allelic variants

Abstract

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”).

1 . A method for detecting a first allelic variant and a second allelic variant of a target sequence in a nucleic acid sample suspected of comprising at least two allelic variants of the target sequence, the method comprising:
 a) performing a multiplex pre-amplification reaction on the nucleic acid sample, the multiplex pre-amplification reaction including a first allele-specific primer, a second allele-specific primer and at least one reverse locus-specific primer,
 wherein the first allele-specific primer includes a first allele-specific nucleotide portion that is complementary to the first allelic variant of the target sequence and the second allele-specific primer includes a second allele-specific nucleotide portion that is complementary to the second allelic variant of the target sequence; 
   b) forming a first reaction mixture by combining:
 i) the pre-amplified nucleic acid sample; 
 ii) the first allele-specific primer; 
 iii) a first allele-specific blocker probe that is complementary to a first region of the target sequence comprising the second allelic variant, wherein said first region encompasses a position corresponding to a binding position of the first allele-specific nucleotide portion of the first allele-specific primer, and wherein the first allele-specific blocker probe comprises a first minor groove binder; 
 iv) a first locus-specific primer that is complementary to a region of the target sequence that is 3′ from the first allelic variant and on an opposite strand; and 
 v) a first detector probe; 
   b) carrying out a first single-plex amplification reaction on the first reaction mixture using the first locus-specific primer and the first allele-specific primer to form a first amplicon;   c) detecting the first amplicon by detecting a change in a detectable property of the first detector probe, thereby detecting the first allelic variant of the target sequence in the nucleic acid sample;   d) forming a second reaction mixture by combining:
 i) the pre-amplified nucleic acid sample; 
 ii) the second allele-specific primer; 
 iii) a second allele-specific blocker probe that is complementary to a second region of the target sequence comprising the first allelic variant, wherein said second region encompasses a position corresponding to a binding position of the second allele-specific nucleotide portion of the second allele-specific primer, and wherein the second allele-specific blocker probe comprises a second minor groove binder; 
 iv) a second locus-specific primer that is complementary to a region of the target sequence that is 3′ from the second allelic variant and on an opposite strand; and 
 v) a second detector probe; 
   e) carrying out a second single-plex amplification reaction on the second reaction mixture using the second allele-specific primer and the second locus-specific primer, to form a second amplicon; and   f) detecting the second amplicon by detecting a change in a detectable property of the second detector probe, thereby detecting the second allelic variant of the target sequence in the nucleic acid sample.   
     
     
         2 . The method of  claim 1 , further comprising using the change in the detectable property of the first detector probe to quantitate the first allelic variant. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , further comprising comparing the change in the detectable property of the first detector probe in the first reaction mixture to the change in the detectable property of the second detector probe in the second reaction mixture. 
     
     
         5 . The method of  claim 1 , wherein at least one of the first allele-specific primer, the second allele-specific primer, the first allele-specific blocker probe or the second allele-specific blocker probe comprises at least one modified base. 
     
     
         6 . The method of  claim 5 , wherein said modified base is an 8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A), cytosine (C), guanine (G), or thymine (T). 
     
     
         7 . The method of  claim 5 , wherein said modified base is a locked nucleic acid (LNA) base. 
     
     
         8 . The method of  claim 5 , wherein said modified base is a fdU or iso dC base. 
     
     
         9 . The method of  claim 5 , wherein said modified base is any modified base that increases the Tm between matched and mismatched target sequences or nucleotides. 
     
     
         10 . The method of  claim 5 , wherein said modified base is located at (a) the 3′-end, (b) the 5′-end, (c) at an internal position or at any combination of (a), (b) or (c) within at least one of the first allele-specific primer, the second allele-specific primer, the first allele-specific blocker probe or the second allele-specific blocker probe. 
     
     
         11 . The method of  claim 5 , wherein the specificity of said detecting is improved by at least 2 fold when said modified base is included in at least one of the first allele-specific primer, the second allele-specific primer, the first allele-specific blocker probe or the second allele-specific blocker probe as compared to when it is not. 
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 1 , wherein the specificity of detecting the first amplicon and/or the second amplicon is improved by at least 2 fold as compared to the specificity of detecting an allelic variant in a nucleic acid sample using ASB-PCR methods. 
     
     
         14 . The method of  claim 1 , wherein the first single-plex and/or second single-plex amplification reaction comprises a 2-stage cycling protocol. 
     
     
         15 . The method of  claim 14 , wherein the number of cycles in the first stage of said 2-stage cycling protocol comprises fewer cycles than the number of cycles used in the second stage. 
     
     
         16 . The method of  claim 14 , wherein said number of cycles in the first stage is about 90% fewer cycles than said number of cycles in the second stage. 
     
     
         17 . The method of  claim 14 , wherein said number of cycles in the first stage is between 3-7 cycles and said number of cycles in the second stage is between 42-48 cycles. 
     
     
         18 . The method of  claim 14 , wherein a first annealing/extension temperature used during the first cycling stage of said 2-stage cycling protocol is between 1-3 2 C lower than a second annealing/extension temperature used during the second stage. 
     
     
         19 . The method of  claim 14 , wherein a first annealing/extension temperature used during the first cycling stage of said 2-stage cycling protocol is between 56-59° C. and a second annealing/extension temperature used during said second stage is between 60-62° C. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein said multiplex amplification reaction further comprises a plurality of allele-specific blocker probes. 
     
     
         24 . The method of  claim 1 , wherein said multiplex amplification reaction is carried out for a number of cycles suitable to keep the reaction within the linear phase of amplification. 
     
     
         25 - 46 . (canceled) 
     
     
         47 . The method of  claim 1 , further comprising using the change in the detectable property of the second detector probe to quantitate the second allelic variant.